1. Endoglin
Endoglin is a homodimeric membrane glycoprotein which is expressed at high levels in proliferating vascular endothelial cells (Burrows et al., 1995, Clin. Cancer Res. 1:1623-1634). Thus, endoglin is primarily a proliferation-associated marker for endothelial cells undergoing active angiogenesis. However, there is some expression of endoglin by the vascular endothelium of normal tissues (Burrows et al., supra; Wang et al., 1993, Int. J. Cancer 54:363-370). Recently, human endoglin was determined to specifically bind transforming growth factor-.beta. (TGF-.beta.), and the deduced amino acid sequence of endoglin showed strong homology to .beta.-glycan, a type of TGF-.beta. receptor.
Murine endoglin has been characterized as a dimer with molecular size of approximately 180 kilodaltons (kD). Human endoglin exists in two forms; i.e., a smaller 160 kD form and a larger 170 kD form with the difference between the two being found in the cytoplasmic portion of the protein. Endoglin has an extracellular region, a hydrophobic transmembrane region, and a cytoplasmic tail. A comparison of the nucleotide sequence of human endoglin with murine endoglin reveals an identity of about 71 to 72% (St. Jacques et al., 1994, Endocrinol. 134:2645-2657; Ge et al., 1994, Gene 158:2645-2657). However, in the human and murine sequences encoding the transmembrane regions and cytoplasmic regions of endoglin, there is a 93-95% identity. Thus, in the human and murine sequences encoding the extracellular region to which antibody would be directed at the cell surface, there is significantly less identity than 70%. Although the amino acid sequence similarity between human and mouse endoglins appears substantial, the observed amino acid sequence differences in the extracellular regions should be sufficient for generating distinct antigenic epitopes unique to human endoglin or to mouse endoglin. This is because in peptide epitopes, even a subtle variation in the amino acid sequence comprising the epitopes or in the flanking amino acid sequences can markedly influence the immunogenicity of the epitopes (see, e.g., Vijayakrishnan et al., 1997, J. Immunol. 159:1809-1819). For instance, single amino acid substitutions in a peptide can cause marked changes in the immunogenicity of the peptide (Vijayakrishnan et al., 1997, supra). Such changes in a peptide epitope will strongly influence the specificity of mAbs because mAbs define fine specificity.
2. Monoclonal Antibodies to Endoglin
There have been several anti-endoglin monoclonal antibodies ("mAb") previously reported in the art. mAb SN6 is an antibody generated from immunization of mice with glycoprotein mixtures of cell membranes of human leukemia cells (Haruta and Seon, 1986, Proc. Natl. Acad. Sci. 83:7898-7902). It is a murine mAb that recognizes human endoglin. mAb 44G4 is an antibody generated from immunization of mice with whole cell suspensions of human pre-B leukemia cells (Gougos and Letarte, 1988, J. Immunol. 141:1925-1933; 1990, J. Biol. Chem. 265:8361-8364). It is a murine mAb that recognizes human endoglin. mAb MJ7/18 is an antibody generated from immunization of rats with inflamed mouse skins (Ge and Butcher, 1994, supra). It is a mAb that recognizes murine endoglin. mAb Tec-11 is an antibody generated from immunization of mice with human umbilical vein endothelial cells (Burrows et al., 1995, Clin. Cancer Res. 1:1623-1634). It is a murine mAb with reactivity restricted to human endoglin.
By the use of anti-endoglin antibodies and various staining procedures known in the art, it has been determined that endoglin is expressed at moderate levels on human tumor cells such as from human leukemia, including non-T-cell-type (non-T) acute lymphoblastic leukemia (ALL), myelo-monocytic leukemia. In addition, it has been determined that endoglin is expressed at moderate to high levels in endothelial cells contained in tumor-associated vasculatures from human solid tumors, including angiosarcoma, breast carcinoma, cecum carcinoma, colon carcinoma, Hodgkins lymphoma, lymphoma, lung carcinoma, melanoma, osteosarcoma, ovarian carcinoma, parotid tumor, pharyngeal carcinoma, rectosigmoid carcinoma; and human vasculature from placenta, adrenal and lymphoid tissues. A lesser degree (weak) endothelial cell staining for endoglin has been observed in a variety of normal human adult tissue sections from spleen, thymus, kidney, lung and liver.
Increased endoglin expression on vascular endothelial cells has also been reported in pathological conditions involving angiogenesis. Such angiogenesis-associated diseases include most types of human solid tumors, rheumatoid arthritis, stomach ulcers, and chronic inflammatory skin lesions (e.g., psoriasis, dermatitis; Westphal et al., 1993, J. Invest. Dermatol. 100:27-34).
3. Angiogenesis
Angiogenesis is the formation of new capillary blood vessels leading to neovascularization. Angiogenesis is a complex process which includes a series of sequential steps including endothelial cell-mediated degradation of vascular basement membrane and interstitial matrices, migration of endothelial cells, proliferation of endothelial cells, and formation of capillary loops by endothelial cells. Solid tumors are angiogenesis-dependent; i.e., as a small solid tumor reaches a critical diameter, for further growth it needs to elicit an angiogenic response in the surrounding normal tissue. The resultant neovascularization of the tumor is associated with more rapid growth, and local invasion. Further, an increase in angiogenesis is associated with an increased risk of metastasis. Therefore, antiangiogenic therapy to inhibit tumor angiogenesis would suppress or arrest tumor growth and its spread.
In normal physiological processes such as wound healing, angiogenesis is turned off once the process is completed. In contrast, tumor angiogenesis is not self-limiting. Further, in certain pathological (and nonmalignant) processes, angiogenesis is abnormally prolonged. Such angiogenesis-associated diseases include diabetic retinopathy, chronic inflammatory diseases including rheumatoid arthritis, dermatitis, and psoriasis. Antiangiogenic therapy would allow modulation in such angiogenesis-associated diseases having excessive vascularization.
4. Antiangiogenic Therapy and Vascular Targeting Therapy of Human Solid Tumors
The progressive growth of solid tumors beyond clinically occult sizes (e.g., a few mm.sup.3) requires the continuous formation of new blood vessels, a process known as tumor angiogenesis. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels to deliver nutrients and oxygen for the tumor itself to grow. Therefore, either prevention of tumor angiogenesis (antiangiogenic therapy) or selective destruction of tumor's existing blood vessels (vascular targeting therapy) present a strategy directed to preventing or treating solid tumors.
Since a local network of new capillary blood vessels provide routes through which the primary tumor may metastasize to other parts of the body, antiangiogenic therapy should be important in preventing establishment of small solid tumors or in preventing metastasis (See, e.g., Folkman, 1995, Nature Medicine, 1:27-31). On the other hand, the vascular targeting therapy which attacks the existing vasculature is likely to be most effective on large tumors where the vasculature is already compromised (See, e.g., Bicknell and Harris, 1992, Semin. Cancer Biol. 3:399-407). Monoclonal antibodies, and fragments thereof according to the present invention are used as a means of delivering to either existing tumor vasculature or newly forming tumor neovascularization therapeutic compounds in a method of antiangiogenic therapy and vascular targeting therapy (collectively referred to as "antiangiogenic therapy").
5. Mouse Models for Human Disease
A. Athymic Nude or SCID Mouse Model
In the following embodiments used to illustrate the invention, it is important to consider the following concept. The use of athymic nude mice with human tumor xenografts has been validated as a model for the evaluation of chemotherapeutic agents because the model has been shown to reflect the clinical effectiveness of chemotherapeutic agents in original patients treated with these agents; and reflects antitumor effects from the agents, such as tumor regression or inhibition of tumor growth, as consistent with the activity against the corresponding types of clinical cancer (See for example, Neuwalt et al., 1985, Cancer Res. 45:2827-2833; Ovejera et al., 1978, Annals of Clin. and Lab. Science 8:50). SCID mice with human tumor xenografts has also been accepted by those skilled in the art as a model for the evaluation of chemotherapeutic agents.
Monoclonal antibodies are useful for selectively targeting tumors and for selective delivery of anticancer agents to tumor target tissue(s). In that regard, anti-endoglin mAbs may be used to target human tumor vasculature. Athymic nude mice or SCID mice with human tumor xenografts is a model in which may be tested antibody-directed targeting of tumor vasculature in a process of antiangiogenic therapy. The problem, however, is that the neovascularization for human xenografts in the mouse model arises from the (mouse) host's tissues. Thus, the prior art anti-endoglin mAbs, which are restricted to reactivity with either human endoglin or murine endoglin, cannot be used in such mouse models to perform the studies necessary to evaluate the clinical efficacy, pharmacokinetics, and the possibility of adverse side effects of antiangiogenic therapy. Therefore, there is a need for an anti-endoglin mAb which specifically binds to a cross-reactive eptitope shared between endoglin on human and murine endothelial cells, wherein such mAbs are essential for performing animal model studies of human solid tumors.
B. Mouse Models for Angiogenesis-associated Diseases
In the following embodiments used to illustrate the invention, it is important to consider the following concept. The use of mouse models of angiogenesis has been accepted and validated as a models for the evaluation of therapeutic agents because the models have been shown to reflect the clinical parameters characteristic of the respective disease, as well as predictive of the effectiveness of therapeutic agents in patients. These mouse models include, but are not limited to: mouse model for retinal neovascularization (Pierce et al., 1995, Proc. Natl. Acad. Sci. USA 92:905-909); mouse models for rheumatoid arthritis (MRL-lpr/lpr mouse model, Folliard et al., 1992, Agents Actions 36:127-135; mev mouse, Kovarik et al., 1994, J. Autoimmun. 7:575-88); mouse models for angiogenesis (Majewski et al., 1994, Int. J. Cancer 57:81-85; Andrade et al., 1992, Int. J. Exp. Pathol., 73:503-13; Sunderkotter et al., 1991, Am. J. Pathol. 138:931-939); mouse model for dermatitis (Maguire et al., 1982, J. Invest. Dermatol. 79:147-152); and mouse model for psoriasis (Blandon et al., 1985, Arch. Dermatol. Res. 277:121-125; Nagano et al., 1990, Arch. Dermatol. Res. 282:459-462). Thus, the prior art anti-endoglin mAbs, which are restricted to reactivity with either human endoglin or murine endoglin, cannot be used in the mouse model for the respective angiogenesis-associated disease to perform the studies necessary to evaluate the clinical efficacy, pharmacokinetics, and adverse side effects of antiangiogenic therapy in humans. Therefore, there is a need for an anti-endoglin mAb which specifically binds to a cross-reactive eptitope shared between endoglin on human and murine endothelial cells, wherein such mAbs are essential for performing animal model studies of angiogenesis-associated diseases.
Hence, a need still exists for anti-endoglin mAbs which can be used in antiangiogenic therapy of human tumor angiogenesis, and of other human angiogenesis-associated diseases having excessive vascularization, which can be evaluated for clinical efficacy and pharmacokinetics in human xenograft-mouse models, or mouse models of angiogenesis-associated diseases.